Trichomonas vaginalis is a sexually-transmitted, obligate extracellular parasite that colonizes the human urogenital tract. Despite being of critica importance to the parasite's survival relatively little is known about the mechanisms employed to establish an infection and thrive within its host. As an extracellular organism, T. vaginalis must adhere to the epithelial lining of the host's urogenital tract to survive. Despite the importance o T. vaginalis surface proteins as a critical interface for pathogen-host interactions, the identity f the surface proteins involved in these processes remains unknown. The overall goals of this proposal are to characterize two surface proteins that are members of the tetraspanin (TSP) family and TSP1-containing exosomes as an abundant surface protein to determine the roles they play in the interaction of this parasite with its human host. Mammalian TSPs exist as membrane complexes that regulate adhesion, migration, intracellular signaling and motility [and our preliminary data indicate that T. vaginalis TSPs are involved in parasite migration]. In specific aim 1 we will determine role of tetraspanin 6 (TSP6) during host-parasite interaction. Our preliminary data show that TSP6 targets to both the plasma membrane and flagella of the parasite and changes its localization upon exposure to host cells. [Moreover, the loss of its 16 amino acid C-terminal intracellular tail results in loss of flagella localization of TSP6 and reducd parasite migration. We propose to further analyze TSP6 function using a knock down approach to test its role in adherence, migration and cytotoxicity to host cells.] In addition, as TSPs act s molecular scaffolds by forming complexes with other cell surface proteins, we will perform co- immunoprecipitation experiments to identify TSP6 partner proteins. These studies will provide the first molecular analyses of tetraspanin proteins in unicellular parasites and enhance our understanding of T. vaginalis host-pathogen interactions. In specific aim 2 we will characterize TSP1 containing exosomes. TSP1 is present on the surface of the cell and on intracellular multivesicular bodies and [exosomes that are released by the parasite. Purification of exosomes has allowed us to show that parasite exosomes induce cytokine release by host cells. We will determine the protein composition of exosomes and their effect on host:parasite communication using TSP1-overexpressing, TSP1-KD and WT parasites.] In addition to providing information about the role of TSP1 in host:pathogen interactions, these studies are the first to examine T. vaginalis exosomes, a key organelle which may mediate communication between T. vaginalis and its host. This research will be done primarily at Fundacin Instituto de Investigaciones Biotecnolgicas (IIB-INTECH) in Chascomus, Argentina in collaboration with Dr. Natalia de Miguel.